Haemophilus influenzae frequently colonizes the human nasopharynx. Up to 80% of the population harbor this organism as part of their normal flora. Although normally an innocuous inhabitant of the upper respiratory tract, H. influenzae is an opportunistic pathogen. The diseases caused by the organism can be ordered in two groups based on the presence or absence of a capsule.
Encapsulated or typeable organisms, which range from capsule types a-f, can cause systemic infections such as bacteremia, septicemia, and bacterial meningitis. Of the various encapsulated types, H. influenzae type b (Hib) has been associated most often with pathogenesis. The non-encapsulated or non-typeable (NTHi) strains of H. influenzae cause more localized infections, such as chronic bronchitis or otitis media, and rarely cause systemic infections.
There are a number of virulence factors associated with both Hib and NTHi that contribute to their pathogenesis, one of these being the lipooligosaccharide (LOS). LOS is a complex glycolipid containing three main regions: lipid A, core, and a variable branched region. The core region is a conserved structure containing a phosphorylated 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) residue linked to three heptose residues, while the variable branched region contains a heterogeneous mix of hexoses and N-acetylhexosamines as well as other factors, such as phosphoethanolamine (PEA), phosphorylcholine, and N-acetylneuraminic acid (NeuAc, also known as sialic acid or SA).
LOS sialylation is believed to influence aspects of susceptibility of a particular bacterial organism to the defense mechanism of a host, e.g., resistance to phagocytosis by neutrophils, the ability of a bacterium to invade a host cell, as well as resistance to the antibody-complement dependent bactericidal effect of serum. The sialylation of LOS is catalyzed by enzymes referred to as sialyltransferases, which are glycosyltransferases. A sialyltransferase catalyzes the transfer of sialic acid (SA) to terminal portions of carbohydrate groups found, for example, on glycolipids and oligosaccharides.
Glycosyltransferases, including sialyltransferases, can be used in vitro to prepare oligosaccharides and polysaccharides (see, e.g., U.S. Pat. Nos. 5,180,674 and 6,210,933). The advantage of biosynthesis with glycosyltransferases is that the glycosidic linkages formed by enzymes are highly stereo and regio-specific. However, each enzyme catalyzes linkage of specific sugar residues to other specific acceptor molecules. Thus, synthesis of a desired oligosaccharide is limited by the availability of glycosyltransferases (see, Roth, International Patent Publication No. WO 93/13198, published Jul. 8, 1993).
There remains a need in the art for the identification and isolation of bacterial sialyltransferases, in particular, sialyltransferases from H. influenzae. Moreover, there remains a need in the art for a method of producing sialylated H. influenzae lipooligosaccharides, e.g., for the production of vaccines against H. influenzae. 